Electronic control of fluidic species

ABSTRACT

Various aspects of the present invention relate to the control and manipulation of fluidic species, for example, in microfluidic systems. In one aspect, the invention relates to systems and methods for making droplets of fluid surrounded by a liquid, using, for example, electric fields, mechanical alterations, the addition of an intervening fluid, etc. In some cases, the droplets may each have a substantially uniform number of entities therein. For example, 95% or more of the droplets may each contain the same number of entities of a particular species. In another aspect, the invention relates to systems and methods for dividing a fluidic droplet into two droplets, for example, through charge and/or dipole interactions with an electric field. The invention also relates to systems and methods for fusing droplets according to another aspect of the invention, for example, through charge and/or dipole interactions. In some cases, the fusion of the droplets may initiate or determine a reaction. In a related aspect of the invention, systems and methods for allowing fluid mixing within droplets to occur are also provided. In still another aspect, the invention relates to systems and methods for sorting droplets, e.g., by causing droplets to move to certain regions within a fluidic system. Examples include using electrical interactions (e.g., charges, dipoles, etc.) or mechanical systems (e.g., fluid displacement) to sort the droplets. In some cases, the fluidic droplets can be sorted at relatively high rates, e.g., at about 10 droplets per second or more. Another aspect of the invention provides the ability to determine droplets, or a component thereof, for example, using fluorescence and/or other optical techniques (e.g., microscopy), or electric sensing techniques such as dielectric sensing.

RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationSerial No. PCT/US2004/027912, filed Aug. 27, 2004, which claims priorityto U.S. Provisional Patent Application Ser. No. 60/498,091, filed Aug.27, 2003, entitled “Electronic Control of Fluidic Species,” by Link, etal., all incorporated herein by reference.

FIELD OF INVENTION

The present invention generally relates to systems and methods for thecontrol of fluidic species and, in particular, to systems and methodsfor the electronic control of fluidic species.

BACKGROUND

The manipulation of fluids to form fluid streams of desiredconfiguration, discontinuous fluid streams, droplets, particles,dispersions, etc., for purposes of fluid delivery, product manufacture,analysis, and the like, is a relatively well-studied art. For example,highly monodisperse gas bubbles, less than 100 microns in diameter, havebeen produced using a technique referred to as capillary flow focusing.In this technique, gas is forced out of a capillary tube into a bath ofliquid, the tube is positioned above a small orifice, and thecontraction flow of the external liquid through this orifice focuses thegas into a thin jet which subsequently breaks into roughly equal-sizedbubbles via capillary instability. In a related technique, a similararrangement can be used to produce liquid droplets in air.

An article entitled “Generation of Steady Liquid Microthreads andMicron-Sized Monodisperse Sprays and Gas Streams,” Phys. Rev. Lett.,80:2, Jan. 12, 1998 (Ganan-Calvo) describes formation of a microscopicliquid thread by a laminar accelerating gas stream, giving rise to afine spray. An articled entitled “Dynamic Pattern Formation in aVesicle-Generating-Microfluidic Device,” Phys. Rev. Lett., 86:18, Apr.30, 2001 (Thorsen, et al.) describes formation of a discontinuous waterphase in a continuous oil phase via microfluidic cross-flow byintroducing water, at a “T” junction between two microfluidic channels,into flowing oil.

U.S. Pat. No. 6,120,666, issued Sep. 19, 2000, describes amicrofabricated device having a fluid focusing chamber for spatiallyconfining first and second sample fluid streams for analyzingmicroscopic particles in a fluid medium, for example, in biologicalfluid analysis. U.S. Pat. No. 6,116,516, issued Sep. 12, 2000, describesformation of a capillary microjet, and formation of a monodisperseaerosol via disassociation of the microjet. U.S. Pat. No. 6,187,214,issued Feb. 13, 2001, describes atomized particles in a size range offrom about 1 to about 5 microns, produced by the interaction of twoimmiscible fluids. U.S. Pat. No. 6,248,378, issued Jun. 19, 2001,describes production of particles for introduction into food using amicrojet and a monodisperse aerosol formed when the microjetdissociates.

Microfluidic systems have been described in a variety of contexts,typically in the context of miniaturized laboratory (e.g., clinical)analysis. Other uses have been described as well. For example,International Patent Publication No. WO 01/89789, published Nov. 29,2001 by Anderson, et al., describes multi-level microfluidic systemsthat can be used to provide patterns of materials, such as biologicalmaterials and cells, on surfaces. Other publications describemicrofluidic systems including valves, switches, and other components.

While significant advances have been made in dynamics at the macro- ormicrofluidic scale, improved techniques and the results of thesetechniques are needed.

SUMMARY OF THE INVENTION

The present invention relates to systems and methods for the electroniccontrol of fluidic species. The subject matter of this inventioninvolves, in some cases, interrelated products, alternative solutions toa particular problem, and/or a plurality of different uses of one ormore systems and/or articles.

In one aspect, the invention provides a method. In one set ofembodiments, the method is a method of combining at least two species ina controlled manner in a microfluidic system. The method includes actsof providing a series of droplets flowing in a microfluidic system;selecting a first droplet from the series of droplets and separating thefirst droplet from at least some other droplets in the series ofdroplets (where the first droplet has a maximum cross-sectionaldimension of less than about 100 microns and contains a first chemical,biological, or biochemical species), providing a second droplet separatefrom the series of droplets (where the second droplet has a maximumcross-sectional dimension of less than about 100 microns and contains asecond chemical, biological, or biochemical species), selectively urgingthe first droplet and/or the second droplet toward a location wherecoalescence can occur and allowing the first droplet and the seconddroplet to coalesce into one combined droplet, and determining areaction involving at least the first species in the first droplet andthe second species in the second droplet.

The method, according to another set of embodiments, is a method ofsorting droplets in a controlled manner in a microfluidic system. Themethod includes acts of providing a series of droplets flowing in amicrofluidic system, and selecting a first droplet from the series ofdroplets and separating the first droplet from at least some otherdroplets in the series of droplets. In some cases, the first droplet hasa maximum cross-sectional dimension of less than about 100 microns.

In yet another set of embodiments, the method is a method of impartingcharge to one or more droplets in a microfluidic system. The method caninclude acts of providing a droplet in a microfluidic system, impartinga dipole moment to the droplet, and dividing the droplet, while thedipole moment is present, into at least two subdroplets, at least one ofthe subdroplets carrying a charge resulting from the dipole momentimparted to the primary droplet.

In still another set of embodiments, the method is a method of combiningat least two droplets in a microfluidic system. The method includes actsof providing at least two droplets in a microfluidic system, exposingthe droplets to an electric field thereby inducing, in the droplets,dipole moments, and coalescing the at least two droplets into a singledroplet at least in part via droplet-droplet attraction due to theinduced dipole moments.

The method, according to another set of embodiments, includes a step ofproducing a charge of at least about 10-14 C on a first fluid surroundedby a second, liquid fluid. The method, according to still another set ofembodiments, includes a step of applying an electric force of at leastabout 10-9 N on a first fluid surrounded by a second, liquid fluid.

In yet another set of embodiments, in a first fluid comprising firstdroplets and second droplets, the first fluid surrounded by a second,liquid fluid, the method comprises sorting the first and second dropletsat a rate of at least about 10 or at least about 100 droplets/s. Instill another set of embodiments, the method includes steps of providingdroplets of a first fluid surrounded by a second, liquid fluid, wherethe droplets have a ratio of droplets containing a first species todroplets free of the first species, and sorting the droplets to increasethe ratio of droplets containing the first species to droplets free ofthe first species by at least a factor of about 2.

In another set of embodiments, in a first fluid comprising firstdroplets and second droplets where the first fluid is surrounded by asecond, liquid fluid, the method comprises sorting the first and seconddroplets without substantially altering a flowrate of the second, liquidfluid. In yet another set of embodiments, the method includes a step ofdividing a first fluidic droplet, surrounded by a second, liquid fluid,into two droplets using an electric field.

In another aspect, the invention is an article. The article, in one setof embodiments, includes a first fluidic droplet having a charge of atleast about 10-14 C, surrounded by a second, liquid fluid. The article,in another set of embodiments, includes droplets comprising a firstfluid surrounded by a second, liquid fluid, where at least about 90% ofthe droplets each consists of the same number of entities of a species.

In yet another aspect, the invention is an apparatus. According to oneset of embodiments, the apparatus includes a microfluidic channel, andan electric field generator constructed and arranged to generate anelectric field of at least about 1 V/micrometer within the microfluidicchannel.

In still another aspect, the present invention is directed to a methodof making one or more of the embodiments described herein. In yetanother aspect, the present invention is directed to a method of usingone or more of the embodiments described herein. In still anotheraspect, the present invention is directed to a method of promoting oneor more of the embodiments described herein.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more applications incorporated by reference include conflictingand/or inconsistent disclosure with respect to each other, then thelater-filed application shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For the purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIGS. 1A and 1B illustrate the splitting of droplets in accordance withone embodiment of the invention;

FIGS. 2A and 2B illustrate an apparatus in accordance with an embodimentof the invention, before the application of an electric field thereto;

FIGS. 3A and 3B illustrate the apparatus of FIGS. 2A and 2B after theapplication of an electric field thereto;

FIGS. 4A and 4B illustrate the apparatus of FIGS. 2A and 2B after theapplication of a reversed electric field thereto;

FIG. 5 is a schematic diagram of droplet splitting, in accordance withone embodiment of the invention;

FIGS. 6A and 6B are schematic diagrams of additional embodiments of theinvention;

FIGS. 7A and 7B illustrate the formation of droplets in accordance withan embodiment of the invention;

FIGS. 8A-8F illustrate the sorting and/or splitting of droplets inaccordance with one embodiment of the invention;

FIGS. 9A-9D illustrate the sorting and/or splitting of droplets inaccordance with another embodiment of the invention;

FIGS. 10A-10D illustrate the sorting of fluidic droplets according toanother embodiment of the invention;

FIGS. 11A-1IC illustrate the sorting of fluidic droplets according toyet another embodiment of the invention;

FIGS. 12A-12J illustrate fluidic mixing in droplets having two or morefluid regions, according to one embodiment of the invention;

FIGS. 13A-13D illustrate uncharged and charged droplets in channels,according to certain embodiments of the invention;

FIGS. 14A-14C illustrate various embodiments of the invention comprisingalternating droplets of a first fluid and of a second fluid; and

FIGS.: 15A-15E illustrate an example of a device featuring variousembodiments of the invention.

DETAILED DESCRIPTION

The present invention generally relates to the control and manipulationof fluidic species, typically surrounded by a liquid (e.g., suspended).Various aspects of the invention relate to forming fluidic droplets,splitting droplets into multiple droplets, creating charges on droplets,inducing dipoles in droplets, causing droplets to fuse or coalesce,causing mixing to occur within droplets, sorting and/or separatingdroplets, and/or sensing and/or determining droplets and/or componentswithin the droplets. Combinations of these and/or other systems andmethods of controlling and manipulating of fluidic species are alsoenvisioned, for example, systems and methods as disclosed in U.S.Provisional Patent Application Ser. No. 60/498,091, filed Aug. 27, 2003,by Link, et. al.; U.S. Provisional Patent Application Ser. No.60/392,195, filed Jun. 28, 2002, by Stone, et. al.; U.S. ProvisionalPatent Application Ser. No. 60/424,042, filed Nov. 5, 2002, by Link, etal.; U.S. Pat. No. 5,512,131, issued Apr. 30, 1996 to Kumar, et al.;International Patent Publication WO 96/29629, published Jun. 26, 1996 byWhitesides, et al.; U.S. Pat. No. 6,355,198, issued Mar. 12, 2002 toKim, et al.; International Patent Application Serial No.:PCT/US01/16973, filed May 25, 2001 by Anderson, et al., published as WO01/89787 on Nov. 29, 2001; International Patent Application Serial No.PCT/US03/20542, filed Jun. 30, 2003 by Stone, et al., published as WO2004/002627 on Jan. 8, 2004; International Patent Application Serial No.PCT/US2004/010903, filed Apr. 9, 2004 by Link, et al.; and U.S.Provisional Patent Application Ser. No. 60/461,954, filed Apr. 10, 2003,by Link, et al.; each of which is incorporated herein by reference.

In various aspects of the invention, a fluidic system as disclosedherein may include a droplet formation system, a sensing system, acontroller, and/or a droplet sorting and/or separation system, or anycombination of these systems. Such systems and methods may be positionedin any suitable order, depending on a particular application, and insome cases, multiple systems of a given type may be used, for example,two or more droplet formation systems, two or more droplet separationsystems, etc. As examples of arrangements, systems of the invention canbe arranged to form droplets, to dilute fluids, to control theconcentration of species within droplets, to sort droplets to selectthose with a desired concentration of species or entities (e.g.,droplets each containing one molecule of reactant), to fuse individualdroplets to cause reaction between species contained in the individualdroplets, to determine reaction(s) and/or rates of reaction(s) in one ormore droplets, etc. Many other arrangements can be practiced inaccordance with the invention.

Droplet Production/Formation

One aspect of the invention relates to systems and methods for producingdroplets of fluid surrounded by a liquid. The fluid and the liquid maybe essentially immiscible in many cases, i.e., immiscible on a timescale of interest (e.g., the time it takes a fluidic droplet to betransported through a particular system or device). In certain cases,the droplets may each be substantially the same shape or size, asfurther described below. The fluid may also contain other species, forexample, certain molecular species (e.g., as further discussed below),cells, particles, etc.

In one set of embodiments, electric charge may be created on a fluidsurrounded by a liquid, which may cause the fluid to separate intoindividual droplets within the liquid. In some embodiments, the fluidand the liquid may be present in a channel, e.g., a microfluidicchannel, or other constricted space that facilitates application of anelectric field to the fluid (which may be “AC” or alternating current,“DC” or direct current etc.), for example, by limiting movement of thefluid with respect to the liquid. Thus, the fluid can be present as aseries of individual charged and/or electrically inducible dropletswithin the liquid. In one embodiment, the electric force exerted on thefluidic droplet may be large enough to cause the droplet to move withinthe liquid. In some cases, the electric force exerted on the fluidicdroplet may be used to direct a desired motion of the droplet within theliquid, for example, to or within a channel or a microfluidic channel(e.g., as further described herein), etc. As one example, in apparatus 5in FIG. 3A, droplets 15 created by fluid source 10 can be electricallycharged using an electric filed created by electric field generator 20.

Electric charge may be created in the fluid within the liquid using anysuitable technique, for example, by placing the fluid within an electricfield (which may be AC, DC, etc.), and/or causing a reaction to occurthat causes the fluid to have an electric charge, for example, achemical reaction, an ionic reaction, a photocatalyzed reaction, etc. Inone embodiment, the fluid is an electrical conductor. As used herein, a“conductor” is a material having a conductivity of at least about theconductivity of 18 megohm (MOhm or) water. The liquid surrounding thefluid may have a conductivity less than that of the fluid. For instance,the liquid may be an insulator, relative to the fluid, or at least a“leaky insulator,” i.e., the liquid is able to at least partiallyelectrically insulate the fluid for at least a short period of time.Those of ordinary skill in the art will be able to identify theconductivity of fluids. In one non-limiting embodiment, the fluid may besubstantially hydrophilic, and the liquid surrounding the fluid may besubstantially hydrophobic.

In some embodiments, the charge created on the fluid (for example, on aseries of fluidic droplets) may be at least about 10-22 C/micrometer3.In certain cases, the charge may be at least about 10-21 C/micrometer3,and in other cases, the charge may be at least about 10-20C/micrometer3, at least about 10-19 C/micrometer3, at least about 10-18C/micrometer3, at least about 10-17 C/micrometer3, at least about 10-16C/micrometer3, at least about 10-15 C/micrometer3, at least about 10-14C/micrometer3, at least about 10-13 C/micrometer3, at least about 10-12C/micrometer3, at least about 10-11 C/micrometer3, at least about 10-10C/micrometer3, or at least about 10-9 C/micrometer3 or more. In certainembodiments, the charge created on the fluid may be at least about 10-21C/micrometer2, and in some cases, the charge may be at least about 10-20C/micrometer2, at least about 10-19 C/micrometer2, at least about 10-18C/micrometer2, at least about 10-17 C/micrometer2, at least about 10-16C/micrometer2, at least about 10-15 C/micrometer2, at least about 10-14C/micrometer2, or at least about 10-13 C/micrometer2 or more. In otherembodiments, the charge may be at least about 10-14 C/droplet, and, insome cases, at least about 10-13 C/droplet, in other cases at leastabout 10-12 C/droplet, in other cases at least about 10-11 C/droplet, inother cases at least about 10-10 C/droplet, or in still other cases atleast about 10-9 C/droplet.

The electric field, in some embodiments, is generated from an electricfield generator, i.e., a device or system able to create an electricfield that can be applied to the fluid. The electric field generator mayproduce an AC field (i.e., one that varies periodically with respect totime, for example, sinusoidally, sawtooth, square, etc.), a DC field(i.e., one that is constant with respect to time), a pulsed field, etc.The electric field generator may be constructed and arranged to createan electric field within a fluid contained within a channel or amicrofluidic channel. The electric field generator may be integral to orseparate from the fluidic system containing the channel or microfluidicchannel, according to some embodiments. As used herein, “integral” meansthat portions of the components integral to each other are joined insuch a way that the components cannot be in manually separated from eachother without cutting or breaking at least one of the components.

Techniques for producing a suitable electric field (which may be AC, DC,etc.) are known to those of ordinary skill in the art. For example, inone embodiment, an electric field is produced by applying voltage acrossa pair of electrodes, which may be positioned on or embedded within thefluidic system (for example, within a substrate defining the channel ormicrofluidic channel), and/or positioned proximate the fluid such thatat least a portion of the electric field interacts with the fluid. Theelectrodes can be fashioned from any suitable electrode material ormaterials known to those of ordinary skill in the art, including, butnot limited to, silver, gold, copper, carbon, platinum, copper,tungsten, tin, cadmium, nickel, indium tin oxide (“ITO”), etc., as wellas combinations thereof. In some cases, transparent or substantiallytransparent electrodes can be used. In certain embodiments, the electricfield generator can be constructed and arranged (e.g., positioned) tocreate an electric field applicable to the fluid of at least about 0.01V/micrometer, and, in some cases, at least about 0.03 V/micrometer, atleast about 0.05 V/micrometer, at least about 0.08 V/micrometer, atleast about 0.1 V/micrometer, at least about 0.3 V/micrometer, at leastabout 0.5 V/micrometer, at least about 0.7 V/micrometer, at least about1 V/micrometer, at least about 1.2 V/micrometer, at least about 1.4V/micrometer, at least about 1.6 V/micrometer, or at least about 2V/micrometer. In some embodiments, even higher electric fieldintensities may be used, for example, at least about 2 V/micrometer, atleast about 3 V/micrometer, at least about 5 V/micrometer, at leastabout 7 V/micrometer, or at least about 10 V/micrometer or more.

In some embodiments, an electric field may be applied to fluidicdroplets to cause the droplets to experience an electric force. Theelectric force exerted on the fluidic droplets may be, in some cases, atleast about 10-16 N/micrometer3. In certain cases, the electric forceexerted on the fluidic droplets may be greater, e.g., at least about10-15 N/micrometer3, at least about 10-14 N/micrometer3, at least about10-13 N/micrometer3, at least about 10-12 N/micrometer3, at least about10-11 N/micrometer3, at least about 10-10 N/micrometer3, at least about10-9 N/micrometer3, at least about 10-8 N/micrometer3, or at least about10-7 N/micrometer3 or more. In other embodiments, the electric forceexerted on the fluidic droplets, relative to the surface area of thefluid, may be at least about 10-15 N/micrometer2, and in some cases, atleast about 10-14 N/micrometer2, at least about 10-13 N/micrometer2, atleast about 10-12 N/micrometer2, at least about 10-11 N/micrometer2, atleast about 10-10 N/micrometer2, at least about 10-9 N/micrometer2, atleast about 10-8 N/micrometer2, at least about 10-7 N/micrometer2, or atleast about 10-6 N/micrometer2 or more. In yet other embodiments, theelectric force exerted on the fluidic droplets may be at least about10-9 N, at least about 10-8 N, at least about 10-7 N, at least about10-6 N, at least about 10-5 N, or at least about 10-4 N or more in somecases.

In some embodiments of the invention, systems and methods are providedfor at least partially neutralizing an electric charge present on afluidic droplet, for example, a fluidic droplet having an electriccharge, as described above. For example, to at least partiallyneutralize the electric charge, the fluidic droplet may be passedthrough an electric field and/or brought near an electrode, e.g., usingtechniques such as those described herein. Upon exiting of the fluidicdroplet from the electric field (i.e., such that the electric field nolonger has a strength able to substantially affect the fluidic droplet),and/or other elimination of the electric field, the fluidic droplet maybecome electrically neutralized, and/or have a reduced electric charge.

In another set of embodiments, droplets of fluid can be created from afluid surrounded by a liquid within a channel by altering the channeldimensions in a manner that is able to induce the fluid to formindividual droplets. The channel may, for example, be a channel thatexpands relative to the direction of flow, e.g., such that the fluiddoes not adhere to the channel walls and forms individual dropletsinstead, or a channel that narrows relative to the direction of flow,e.g., such that the fluid is forced to coalesce into individualdroplets. One example is shown in FIG. 7A, where channel 510 includes aflowing fluid 500 (flowing downwards), surrounded by liquid 505. Channel510 narrows at location 501, causing fluid 500 to form a series ofindividual fluidic droplets 515. In other embodiments, internalobstructions may also be used to cause droplet formation to occur. Forinstance, baffles, ridges, posts, or the like may be used to disruptliquid flow in a manner that causes the fluid to coalesce into fluidicdroplets.

In some cases, the-channel dimensions may be altered with respect totime (for example, mechanically or electromechanically, pneumatically,etc.) in such a manner as to cause the formation of individual fluidicdroplets to occur. For example, the channel may be mechanicallycontracted (“squeezed”) to cause droplet formation, or a fluid streammay be mechanically disrupted to cause droplet formation, for example,through the use of moving baffles, rotating blades, or the like. As anon-limiting example, in FIG. 7B, fluid 500 flows through channel 510 ina downward direction. Fluid 500 is surrounded by liquid 505.Piezoelectric devices 520 positioned near or integral to channel 510 maythen mechanically constrict or “squeeze” channel 510, causing fluid 500to break up into individual fluidic droplets 515.

In yet another set of embodiments, individual fluidic droplets can becreated and maintained in a system comprising three essentially mutuallyimmiscible fluids (i.e., immiscible on a time scale of interest), whereone fluid is a liquid carrier, and the second fluid and the third fluidalternate as-individual fluidic droplets within the liquid carrier. Insuch a system, surfactants are not necessarily required to ensureseparation of the fluidic droplets of the second and third fluids. As anexample, with reference to FIG. 14A, within channel 700, a first fluid701 and a second fluid 702 are each carried within liquid carrier 705.First fluid 701 and second fluid 702 alternate as a series ofalternating, individual droplets, each carried by liquid carrier 705within channel 700. As the first fluid, the second fluid, and the liquidcarrier are all essentially mutually immiscible, any two of the fluids(or all three fluids) can come into contact without causing dropletcoalescence to occur. A photomicrograph of an example of such a systemis shown in FIG. 14B, illustrating first fluid 701 and second fluid 702,present as individual, alternating droplets, each contained withinliquid carrier 705.

One example of a system involving three essentially mutually immisciblefluids is a silicone oil, a mineral oil, and an aqueous solution (i.e.,water, or water containing one or more other species that are dissolvedand/or suspended therein, for example, a salt solution, a salinesolution, a suspension of water containing particles or cells, or thelike). Another example of a system is a silicone oil, a fluorocarbonoil, and an aqueous solution. Yet another example of a system is ahydrocarbon oil (e.g., hexadecane), a fluorocarbon oil, and an aqueoussolution. In these examples, any of these fluids may be used as theliquid carrier. Non-limiting examples of suitable fluorocarbon oilsinclude octadecafluorodecahydronaphthalene:

or 1-(1,2,2,3,3,4,4,5,5,6,6-undecafluorocyclohexyl)ethanol:

A non-limiting example of such a system is illustrated in FIG. 14B. Inthis figure, fluidic network 710 includes a channel containing liquidcarrier 705, and first fluid 701 and second fluid 702. Liquid carrier705 is introduced into fluidic network 710 through inlet 725, whilefirst fluid 701 is introduced through inlet 721, and second fluid 702 isintroduced through inlet 722. Channel 716 within fluidic network 710contains liquid carrier 715 introduced from inlet 725. Initially, firstfluid 701 is introduced into liquid carrier 705, forming fluidicdroplets therein. Next, second fluid 702 is introduced into liquid 705,forming fluidic droplets therein that are interspersed with the fluidicdroplets containing first fluid 701. Thus, upon reaching channel 717,liquid carrier 705 contains a first set of fluidic droplets containingfirst fluid 701, interspersed with a second set of fluidic dropletscontaining second fluid 702. In the embodiment illustrated, channel 706optionally comprises a series of bends, which may allow mixing to occurwithin each of the fluidic droplets, as further discussed below.However, it should be noted that in this embodiment, since first fluid701 and second fluid 702 are essentially immiscible, significant fusionand/or mixing of the droplets containing first fluid 701 with thedroplets containing second fluid 702 is not generally expected.

Other examples of the production of droplets of fluid surrounded by aliquid are described in International Patent Application Serial No.PCT/US2004/010903, filed Apr. 9, 2004 by Link, et al. and InternationalPatent Application Serial No. PCT/US03/20542, filed Jun. 30, 2003 byStone, et al., published as WO 2004/002627 on Jan. 8, 2004, eachincorporated herein by reference.

In some embodiments, the fluidic droplets may each be substantially thesame shape and/or size. The shape and/or size can be determined, forexample, by measuring the average diameter or other characteristicdimension of the droplets. The term “determining,” as used herein,generally refers to the analysis or measurement of a species, forexample, quantitatively or qualitatively, and/or the detection of thepresence or absence of the species. “Determining” may also refer to theanalysis or measurement of an interaction between two or more species,for example, quantitatively or qualitatively, or by detecting thepresence or absence of the interaction. Examples of suitable techniquesinclude, but are not limited to, spectroscopy such as infrared,absorption, fluorescence, UV/visible, FTIR (“Fourier Transform InfraredSpectroscopy”), or Raman; gravimetric techniques; ellipsometry;piezoelectric measurements; immunoassays; electrochemical measurements;optical measurements such as optical density measurements; circulardichroism; light scattering measurements such as quasielectric lightscattering; polarimetry; refractometry; or turbidity measurements.

The “average diameter” of a plurality or series of droplets is thearithmetic average of the average diameters of each of the droplets.Those of ordinary skill in the art will be able to determine the averagediameter (or other characteristic dimension) of a plurality or series ofdroplets, for example, using laser light scattering, microscopicexamination, or other known techniques. The diameter of a droplet, in anon-spherical droplet, is the mathematically-defined average diameter ofthe droplet, integrated across the entire surface. The average diameterof a droplet (and/or of a plurality or series of droplets) may be, forexample, less than about 1 mm, less than about 500 micrometers, lessthan about 200 micrometers, less than about 100 micrometers, less thanabout 75 micrometers, less than about 50 micrometers, less than about 25micrometers, less than about 10 micrometers, or less than about 5micrometers in some cases. The average diameter may also be at leastabout 1 micrometer, at least about 2 micrometers, at least about 3micrometers, at least about 5 micrometers, at least about 10micrometers, at least about 15 micrometers, or at least about 20micrometers in certain cases.

In certain embodiments of the invention, the fluidic droplets maycontain additional entities, for example, other chemical, biochemical,or biological entities (e.g., dissolved or suspended in the fluid),cells, particles, gases, molecules, or the like. In some cases, thedroplets may each be substantially the same shape or size, as discussedabove. In certain instances, the invention provides for the productionof droplets consisting essentially of a substantially uniform number ofentities of a species therein (i.e., molecules, cells, particles, etc.).For example, about 90%, about 93%, about 95%, about 97%, about 98%, orabout 99%, or more of a plurality or series of droplets may each containthe same number of entities of a particular species. For instance, asubstantial number of fluidic droplets produced, e.g., as describedabove, may each contain 1 entity, 2 entities, 3 entities, 4 entities, 5entities, 7 entities, 10 entities, 15 entities, 20 entities, 25entities, 30 entities, 40 entities, 50 entities, 60 entities, 70entities, 80 entities, 90 entities, 100 entities, etc., where theentities are molecules or macromolecules, cells, particles, etc. In somecases, the droplets may each independently contain a range of entities,for example, less than 20 entities, less than 15 entities, less than 10entities, less than 7 entities, less than 5 entities, or less than 3entities in some cases. In one set of embodiments, in a liquidcontaining droplets of fluid, some of which contain a species ofinterest and some of which do not contain the species of interest, thedroplets of fluid may be screened or sorted for those droplets of fluidcontaining the species as further described below (e.g., usingfluorescence or other techniques such as those described above), and insome cases, the droplets may be screened or sorted for those droplets offluid containing a particular number or range of entities of the speciesof interest, e.g., as previously described. Thus, in some cases, aplurality or series of fluidic droplets, some of which contain thespecies and some of which do not, may be enriched (or depleted) in theratio of droplets that do contain the species, for example, by a factorof at least about 2, at least about 3, at least about 5, at least about10, at least about 15, at least about 20, at least about 50, at leastabout 100, at least about 125, at least about 150, at least about 200,at least about 250, at least about 500, at least about 750, at leastabout 1000, at least about 2000, or at least about 5000 or more in somecases. In other cases, the enrichment (or depletion) may be in a ratioof at least about 104, at least about 105, at least about 106, at leastabout 107, at least about 108, at least about 109, at least about 1010,at least about 1011, at least about 1012, at least about 1013, at leastabout 1014, at least about 1015, or more. For example, a fluidic dropletcontaining a particular species may be selected from a library offluidic droplets containing various species, where the library may haveabout 105, about 106, about 107, about 108, about 109, about 1010, about1011, about 1012, about 1013, about 1014, about 1015, or more items, forexample, a DNA library, an RNA library, a protein library, acombinatorial chemistry library, etc. In certain embodiments, thedroplets carrying the species may then be fused, reacted, or otherwiseused or processed, etc., as further described below, for example, toinitiate or determine a reaction.

Splitting Droplets

In another aspect, the invention relates to systems and methods forsplitting a fluidic droplet into two or more droplets. The fluidicdroplet may be surrounded by a liquid, e.g., as previously described,and the fluid and the liquid are essentially immiscible in some cases.The two or more droplets created by splitting the original fluidicdroplet may each be substantially the same shape and/or size, or the twoor more droplets may have different shapes and/or sizes, depending onthe conditions used to split the original fluidic droplet. In manycases, the conditions used to split the original fluidic droplet can becontrolled in some fashion, for example, manually or automatically(e.g., with a processor, as discussed below). In some cases, eachdroplet in a plurality or series of fluidic droplets may beindependently controlled. For example, some droplets may be split intoequal parts or unequal parts, while other droplets are not split.

According to one set of embodiments, a fluidic droplet can be splitusing an applied electric field. The electric field may be an AC field,a DC field, etc. The fluidic droplet, in this embodiment, may have agreater electrical conductivity than the surrounding liquid, and, insome cases, the fluidic droplet may be neutrally charged. In someembodiments, the droplets produced from the original fluidic droplet areof approximately equal shape and/or size. In certain embodiments, in anapplied electric field, electric charge may be urged to migrate from theinterior of the fluidic droplet to the surface to be distributedthereon, which may thereby cancel the electric field experienced in theinterior of the droplet. In some embodiments, the electric charge on thesurface of the fluidic droplet may also experience a force due to theapplied electric field, which causes charges having opposite polaritiesto migrate in opposite directions. The charge migration may, in somecases, cause the drop to be pulled apart into two separate fluidicdroplets. The electric field applied to the fluidic droplets may becreated, for example, using the techniques described above, such as witha reaction an electric field generator, etc.

As a non-limiting example, in FIG. 1A, where no electric field isapplied, fluidic droplets 215 contained in channel 230 are carried by asurrounding liquid, which flows towards intersection 240, leading tochannels 250 and 255. In this example, the surrounding liquid flowsthrough channels 250 and 255 at equal flowrates. Thus, at intersection240, fluidic droplets 215 do not have a preferred orientation ordirection, and move into exit channels 250 and 255 with equalprobability due to the surrounding liquid flow. In contrast, in FIG. 1B,while the surrounding liquid flows in the same fashion as FIG. 1A, underthe influence of an applied electric field of 14 V/micrometers, fluidicdroplets 215 are split into two droplets at intersection 240, formingnew droplets 216 and 217. Droplet 216 moves to the left in channel 250,while droplet 217 moves to the right in channel 255.

A schematic of this process can be seen in FIG. 5, where a neutralfluidic droplet 530, surrounded by a liquid 535 in channel 540, issubjected to applied electric field 525, created by electrodes 526 and527. Electrode 526 is positioned near channel 542, while electrode 527is positioned near channel 544. Under the influence of electric field525, charge separation is induced within fluidic droplet 530, i.e., suchthat a positive charge is induced at one end of the droplet, while anegative charge is induced at the other end of the droplet. The dropletmay then split into a negatively charged droplet 545 and a positivelycharged droplet 546, which then may travel in channels 542 and 544,respectively. In some cases, one or both of the electric charges on theresulting charged droplets may also be neutralized, as previouslydescribed.

Other examples of splitting a fluidic droplet into two droplets aredescribed in International Patent Application Serial No.PCT/US2004/010903, filed Apr. 9, 2004 by Link, et al.; U.S. ProvisionalPatent Application Ser. No. 60/498,091, filed Aug. 27, 2003, by Link,et. al.; and International Patent Application Serial No. PCT/US03/20542,filed Jun. 30, 2003 by Stone, et al., published as WO 2004/002627 onJan. 8, 2004, each incorporated herein by reference.

Fusing Droplets

The invention, in yet another aspect, relates to systems and methods forfusing or coalescing two or more fluidic droplets into one droplet. Forexample, in one set of embodiments, systems and methods are providedthat are able to cause two or more droplets (e.g., arising fromdiscontinuous streams of fluid) to fuse or coalesce into one droplet incases where the two or more droplets ordinarily are unable to fuse orcoalesce, for example, due to composition, surface tension, dropletsize, the presence or absence of surfactants, etc. In certainmicrofluidic systems, the surface tension of the droplets, relative tothe size of the droplets, may also prevent fusion or coalescence of thedroplets from occurring in some cases.

In one embodiment, two fluidic droplets may be given opposite electriccharges (i.e., positive and negative charges, not necessarily of thesame magnitude), which may increase the electrical interaction of thetwo droplets such that fusion or coalescence of the droplets can occurdue to their opposite electric charges, e.g., using the techniquesdescribed herein. For instance, an electric field may be applied to thedroplets, the droplets may be passed through a capacitor, a chemicalreaction may cause the droplets to become charged, etc. As an example,as is shown schematically in FIG. 13A, uncharged droplets 651 and 652,carried by a liquid 654 contained within a microfluidic channel 653, arebrought into contact with each other, but the droplets are not able tofuse or coalesce, for instance, due to their size and/or surfacetension. The droplets, in some cases, may not be able to fuse even if asurfactant is applied to lower the surface tension of the droplets.However, if the fluidic droplets are electrically charged with oppositecharges (which can be, but are not necessarily of, the same magnitude),the droplets may be able to fuse or coalesce. For instance, in FIG. 13B,positively charged droplets 655 and negatively charged droplets 656 aredirected generally towards each other such that the electricalinteraction of the oppositely charged droplets causes the droplets tofuse into fused droplets 657.

In another embodiment, the fluidic droplets may not necessarily be givenopposite electric charges (and, in some cases, may not be given anyelectric charge), and are fused through the use of dipoles induced inthe fluidic droplets that causes the fluidic droplets to coalesce. Inthe example illustrated in FIG. 13C, droplets 660 and 661 (which mayeach independently be electrically charged or neutral), surrounded byliquid 665 in channel 670, move through the channel such that they arethe affected by an applied electric field 675. Electric field 675 may bean AC field, a DC field, etc., and may be created, for instance, usingelectrodes 676 and 677, as shown here. The induced dipoles in each ofthe fluidic droplets, as shown in FIG. 13C, may cause the fluidicdroplets to become electrically attracted towards each other due totheir local opposite charges, thus causing droplets 660 and 661 to fuseto produce droplet 663. In FIG. 13D, droplets 660 and 661

It should be noted that, in various embodiments, the two or moredroplets allowed to coalesce are not necessarily required to meet“head-on.” Any angle of contact, so long as at least some fusion of thedroplets initially occurs, is sufficient. As an example, in FIG. 12H,droplets 73 and 74 each are traveling in substantially the samedirection (e.g., at different velocities), and are able to meet andfuse. As another example, in FIG. 121, droplets 73 and 74 meet at anangle and fuse. In FIG. 12J, three fluidic droplets 73, 74 and 68 meetand fuse to produce droplet 79.

Other examples of fusing or coalescing fluidic droplets are described inInternational Patent Application Serial No. PCT/US2004/010903, filedApr. 9, 2004 by Link, et al., incorporated herein by reference.

Mixing within Droplets

In a related aspect, the invention relates to systems and methods forallowing the mixing of more than one fluid to occur within a fluidicdroplet. For example, in various embodiments of the invention, two ormore fluidic droplets may be allowed to fuse or coalesce, as describedabove, and then, within the fused droplet, the two or more fluids fromthe two or more original fluidic droplets may then be allowed to mix. Itshould be noted that when two droplets fuse or coalesce, perfect mixingwithin the droplet does not instantaneously occur. Instead, for example,as is shown in FIG. 12B, the coalesced droplet may initially be formedof a first fluid region 71 (from first droplet 73) and a second fluidregion 72 (from second droplet 74). Thus, in some cases, the fluidregions may remain as separate regions, for example, due to internal“counter-revolutionary” flow within the fluidic droplet (shown in FIG.12G with droplet 968, direction indicated by arrow 977), thus resultingin a non-uniform fluidic droplet 75, as is shown in FIG. 12A.

However, in other cases, the fluid regions within the fluidic dropletmay be allowed to mix, react, or otherwise interact with each other, asis shown in FIG. 7B, resulting in mixed or partially mixed fluidicdroplet 78. The mixing may occur through natural means, for example,through diffusion (e.g., through the interface between the regions),through reaction of the fluids with each other, through fluid flowwithin the droplet (i.e., convection), etc. However, in some cases,mixing of the regions 71 and 72 may be enhanced through certain systemsexternal of the fluidic droplet. For example, the fluidic droplet may bepassed through one or more channels or other systems which cause thedroplet to change its velocity and/or direction of movement. The changeof direction may alter convection patterns within the droplet, causingthe fluids to be at least partially mixed. As an example, in FIG. 12C,droplet 76 may be passed through one or more bends within a channel,causing the fluids within droplet 76 to be at least partially mixed,resulting in droplet 79; or droplet 76 may pass by one or moreobstructions within the channel, etc. As another example, in FIG. 12D,droplet 76 passes through one or more expansion regions 77 within achannel, causing the fluids within droplet 76 to be at least partiallymixed, resulting in droplet 79. In FIG. 12E, droplet 76 passes throughone or more constriction regions 69, causing the fluids within droplet76 to be at least partially mixed, resulting in droplet 79. Combinationsare also possible. For example, in FIG. 12F, droplet 76 passes throughbend 70, expansion region 77, and constriction region 69, causing atleast partial mixing of the fluid regions within the droplet to occur.As yet another example, channel 706 in FIG. 14B contains a series ofbends, which may allow mixing of the fluids within the droplets withinchannel 706 to occur.

In one set of embodiments, a fluid may be injected into a fluidicdroplet, which may cause mixing of the injected fluid with the otherfluids within the fluidic droplet to occur. The fluid may bemicroinjected into the fluidic droplet in some cases, e.g., using amicroneedle or other such device. In other cases, the fluid may beinjected directly into a fluidic droplet using a fluidic channel as thefluidic droplet comes into contact with the fluidic channel. Forinstance, referring now to FIG. 14C, channel 750 contains a carrierfluid 755 containing a series of fluidic droplets 760. Droplet 761 is incontact with fluidic channel 752. A fluid can then be introduced intofluidic droplet 761 through fluidic channel 752, which fluid may be thesame or different than the fluid in fluidic droplet 761.

Other examples of fluidic mixing in droplets are described inInternational Patent Application Serial No. PCT/US2004/010903, filedApr. 9, 2004 by Link, et al., incorporated herein by reference.

Screening/Sorting Droplets

In still another aspect, the invention provides systems and methods forscreening or sorting fluidic droplets in a liquid, and in some cases, atrelatively high rates. For example, a characteristic of a droplet may besensed and/or determined in some fashion (e.g., as further describedbelow), then the droplet may be directed towards a particular region ofthe device, for example, for sorting or screening purposes.

In some embodiments, a characteristic of a fluidic droplet may be sensedand/or determined in some fashion, for example, as described herein(e.g., fluorescence of the fluidic droplet may be determined), and, inresponse, an electric field may be applied or removed from the fluidicdroplet to direct the fluidic droplet to a particular region (e.g. achannel). In some cases, high sorting speeds may be achievable usingcertain systems and methods of the invention. For instance, at leastabout 10 droplets per second may be determined and/or sorted in somecases, and in other cases, at least about 20 droplets per second, atleast about 30 droplets per second, at least about 100 droplets persecond, at least about 200 droplets per second, at least about 300droplets per second, at least about 500 droplets per second, at leastabout 750 droplets per second, at least about 1000 droplets per second,at least about 1500 droplets per second, at least about 2000 dropletsper second, at least about 3000 droplets per second, at least about 5000droplets per second, at least about 7500 droplets per second, at leastabout 10,000 droplets per second, at least about 15,000 droplets persecond, at least about 20,000 droplets per second, at least about 30,000droplets per second, at least about 50,000 droplets per second, at leastabout 75,000 droplets per second, at least about 100,000 droplets persecond, at least about 150,000 droplets per second, at least about200,000 droplets per second, at least about 300,000 droplets per second,at least about 500,000 droplets per second, at least about 750,000droplets per second, at least about 1,000,000 droplets per second, atleast about 1,500,000 droplets per second, at least about 2,000,000 ormore droplets per second, or at least about 3,000,000 or more dropletsper second may be determined and/or sorted in such a fashion.

In one set of embodiments, a fluidic droplet may be directed by creatingan electric charge (e.g., as previously described) on the droplet, andsteering the droplet using an applied electric field, which may be an ACfield, a DC field, etc. As an example, in reference to FIGS. 2-4, anelectric field maybe selectively applied and removed (or a differentelectric field may be applied, e.g., a reversed electric field as shownin FIG. 4A) as needed to direct the fluidic droplet to a particularregion. The electric field may be selectively applied and removed asneeded, in some embodiments, without substantially altering the flow ofthe liquid containing the fluidic droplet. For example, a liquid mayflow on a substantially steady-state basis (i.e., the average flowrateof the liquid containing the fluidic droplet deviates by less than 20%or less than 15% of the steady-state flow or the expected value of theflow of liquid with respect to time, and in some cases, the averageflowrate may deviate less than 10% or less than 5%) or otherpredetermined basis through a fluidic system of the invention (e.g.,through a channel or a microchannel), and fluidic droplets containedwithin the liquid may be directed to various regions, e.g., using anelectric field, without substantially altering the flow of the liquidthrough the fluidic system. As a particular example, in FIGS. 2A, 3A and4A, a liquid containing fluidic droplets 15 flows from fluid source 10,through channel 30 to intersection 40, and exits through channels 50 and55. In FIG. 2A, fluidic droplets 15 are directed through both channels50 and 55, while in FIG. 3A, fluidic droplets 15 are directed to onlychannel 55 and, in FIG. 4A, fluidic droplets 15 are directed to onlychannel 50.

In another set of embodiments, a fluidic droplet may be sorted orsteered by inducing a dipole in the fluidic droplet (which may beinitially charged or uncharged), and sorting or steering the dropletusing an applied electric field. The electric field may be an AC field,a DC field, etc. For example, with reference to FIG. 9A, a channel 540,containing fluidic droplet 530 and liquid 535, divides into channel 542and 544. Fluidic droplet 530 may have an electric charge, or it may beuncharged. Electrode 526 is positioned near channel 542, while electrode527 is positioned near channel 544. Electrode 528 is positioned near thejunction of channels 540, 542, and 544. In FIGS. 9C and 9D, a dipole isinduced in the fluidic droplet using electrodes 526, 527, and/or 528. InFIG. 9C, a dipole is induced in droplet 530 by applying an electricfield 525 to the droplet using electrodes 527 and 528. Due to thestrength of the electric field, the droplet is strongly attracted to theright, into channel 544. Similarly, in FIG. 9D, a dipole is induced indroplet 530 by applying an electric field 525 to the droplet usingelectrodes 526 and 528, causing the droplet to be attracted into channel542. Thus, by applying the proper electric field, droplet 530 can bedirected to either channel 542 or 544 as desired.

In other embodiments, however, the fluidic droplets may be screened orsorted within a fluidic system of the invention by altering the flow ofthe liquid containing the droplets. For instance, in one set ofembodiments, a fluidic droplet may be steered or sorted by directing theliquid surrounding the fluidic droplet into a first channel, a secondchannel, etc. As a non-limiting example, with reference to FIG. 10A,fluidic droplet 570 is surrounded by a liquid 575 in channel 580.Channel 580 divides into three channels 581, 582, and 583. The flow ofliquid 575 can be directed into any of channels 581, 582, and 583 asdesired, for example, using flow-controlling devices known to those ofordinary skill in the art, for example, valves, pumps, pistons, etc.Thus, in FIG. 10B, fluidic droplet 570 is directed into channel 581 bydirecting liquid 575 to flow into channel 581 (indicated by arrows 574);in FIG. 10C, fluidic droplet 570 is directed into channel 582 bydirecting liquid 575 to flow into channel 582 (indicated by arrows 574);and in FIG. 10D, fluidic droplet 570 is directed into channel 583 bydirecting liquid 575 to flow into channel 583 (indicated by arrows 574).

In another set of embodiments, pressure within a fluidic system, forexample, within different channels or within different portions of achannel, can be controlled to direct the flow of fluidic droplets. Forexample, a droplet can be directed toward a channel junction includingmultiple options for further direction of flow (e.g., directed toward abranch, or fork, in a channel defining optional downstream flowchannels). Pressure within one or more of the optional downstream flowchannels can be controlled to direct the droplet selectively into one ofthe channels, and changes in pressure can be effected on the order ofthe time required for successive droplets to reach the junction, suchthat the downstream flow path of each successive droplet can beindependently controlled. In one arrangement, the expansion and/orcontraction of liquid reservoirs may be used to steer or sort a fluidicdroplet into a channel, e.g., by causing directed movement of the liquidcontaining the fluidic droplet. The liquid reservoirs may be positionedsuch that, when activated, the movement of liquid caused by theactivated reservoirs causes the liquid to flow in a preferred direction,carrying the fluidic droplet in that preferred direction. For instance,the expansion of a liquid reservoir may cause a flow of liquid towardsthe reservoir, while the contraction of a liquid reservoir may cause aflow of liquid away from the reservoir. In some cases, the expansionand/or contraction of the liquid reservoir may be combined with otherflow-controlling devices and methods, e.g., as described herein.Non-limiting examples of devices able to cause the expansion and/orcontraction of a liquid reservoir include pistons and piezoelectriccomponents. In some cases, piezoelectric components may be particularlyuseful due to their relatively rapid response times, e.g., in responseto an electrical signal.

As a non-limiting example, in FIG. 11A, fluidic droplet 600 issurrounded by a liquid 605 in channel 610. Channel 610 divides intochannels 611, 612. Positioned in fluidic communication with channels 611and 612 are liquid reservoirs 617 and 618, which may be expanded and/orcontracted, for instance, by piezoelectric components 615 and 616, by apiston (not shown), etc. In FIG. 11B, liquid reservoir 617 has beenexpanded, while liquid reservoir 618 has been contracted. The effect ofthe expansion/contractions of the reservoirs is to cause a net flow ofliquid towards channel 611, as indicated by arrows 603. Thus, fluidicdroplet 600, upon reaching the junction between the channels, isdirected to channel 611 by the movement of liquid 605. The reversesituation is shown in FIG. 11C, where liquid reservoir 617 hascontracted while liquid reservoir 618 has been expanded. A net flow ofliquid occurs towards channel 612 (indicated by arrows 603), causingfluidic droplet 600 to move into channel 612. It should be noted,however, that reservoirs 617 and 618 do not both need to be activated todirect fluidic droplet 600 into channels 611 or 612. For example, in oneembodiment, fluidic droplet 600 may be directed to channel 611 by theexpansion of liquid reservoir 617 (without any alteration of reservoir618), while in another embodiment, fluidic droplet 600 may be directedto channel 611 by the contraction of liquid reservoir 618 (without anyalteration of reservoir 617). In some cases, more than two liquidreservoirs may be used.

In some embodiments, the fluidic droplets may be sorted into more thantwo channels. Non-limiting examples of embodiments of the inventionhaving multiple regions within a fluidic system for the delivery ofdroplets are shown in FIGS. 6A and 6B. Other arrangements are shown inFIGS. 10A-10D. In FIG. 6A, charged droplets 315 in channel 330 may bedirected as desired to any one of exit channels 350, 352, 354, or 356,by applying electric fields to control the movement of the droplets atintersections 340, 341, and 342, using electrodes 321/322, 323/324, and325/326, respectively. In FIG. 6A, droplets 315 are directed to channel354 using applied electric fields 300 and 301, using principles similarto those discussed above. Similarly, in FIG. 6B, charged droplets 415 inchannel 430 can be directed to any one of exit channels 450, 452, 454,456, or 458, by applying electric fields to control the movement of thedroplets at intersections 440, 441, 442, and 443, using electrodes421/422, 423/424, 425/426, and 427/428, respectively. In this figure,droplets 415 are directed to channel 454; of course, the chargeddroplets may be directed to any other exit channel as desired.

In another example, in apparatus 5, as schematically illustrated in FIG.2A, fluidic droplets 15 created by fluid source 10 are positivelycharged due to an applied electric field created using electric fieldgenerator 20, which comprises two electrodes 22, 24. Fluidic droplets 15are directed through channel 30 by a liquid containing the droplets, andare directed towards intersection 40. At intersection 40, the fluidicdroplets do not have a preferred orientation or direction, and move intoexit channels 50 and 55 with equal probability (in this embodiment,liquid drains through both exit channels 50 and 55 at substantiallyequal rates). Similarly, fluidic droplets 115 created by fluid source110 are negatively charged due to an applied electric field createdusing electric field generator 120, which comprises electrodes 122 and124. After traveling through channel 130 towards intersection 140, thefluidic droplets do not have a preferred orientation or direction, andmove into exit channels 150 and 155 with equal probability, as theliquid exits through exit channels 150 and 155 at substantially equalrates. A representative photomicrograph of intersection 140 is shown inFIG. 2B.

In the schematic diagram of FIG. 3A, an electric field 100 of 1.4V/micrometer has been applied to apparatus 5 of FIG. 2A, in a directiontowards the right of apparatus 5. Positively-charged fluidic droplets 15in channel 30, upon reaching intersection 40, are directed to the rightin channel 55 due to the applied electric field 100, while the liquidcontaining the droplets continues to exit through exit channels 50 and55 at substantially equal rates. Similarly, negatively-charged fluidicdroplets 115 in channel 130, upon reaching intersection 140, aredirected to the left in channel 150 due to the applied electric field100, while the liquid fluid continues to exit the device through exitchannels 150 and 155 at substantially equal rates. Thus, electric field100 can be used to direct fluidic droplets into particular channels asdesired. A representative photomicrograph of intersection 140 is shownin FIG. 3B.

FIG. 4A is a schematic diagram of apparatus 5 of FIG. 2A, also with anapplied electric field 100 of 1.4 V/micrometer, but in the oppositedirection (i.e., −1.4 V/micrometer). In this figure, positively-chargedfluidic droplets 15 in channel 30, upon reaching intersection 40, aredirected to the left into channel 50 due to the applied electric field100, while negatively-charged fluidic droplets 115 in channel 130, uponreaching intersection 140, are directed to the right into channel 155due to applied electric field 100. The liquid containing the dropletsexits through exit channels 50 and 55, and 150 and 155, at substantiallyequal rates. A representative photomicrograph of intersection 140 isshown in FIG. 4B.

In some embodiments of the invention, a fluidic droplet may be sortedand/or split into two or more separate droplets, for example, dependingon the particular application. Any of the above-described techniques maybe used to spilt and/or sort droplets. As a non-limiting example, byapplying (or removing) a first electric field to a device (or a portionthereof), a fluidic droplet may be directed to a first region orchannel; by applying (or removing) a second electric field to the device(or a portion thereof), the droplet may be directed to a second regionor channel; by applying a third electric field to the device (or aportion thereof), the droplet may be directed to a third region orchannel; etc., where the electric fields may differ in some way, forexample, in intensity, direction, frequency, duration, etc. In a seriesof droplets, each droplet may be independently sorted and/or split; forexample, some droplets may be directed to one location or another, whileother droplets may be split into multiple droplets directed to two ormore locations.

As one particular example, in FIG. 8A, fluidic droplet 550, surroundingliquid 555 in channel 560 may be directed to channel 556, channel 557,or be split in some fashion between channels 562 and 564. In FIG. 8B, bydirecting surrounding liquid 555 towards channel 562, fluidic droplet550 may be directed towards the left into channel 562; in FIG. 8C, bydirecting surrounding liquid 555 towards channel 564, fluidic droplet550 may be directed towards the right into channel 564, In FIG. 8D, anelectric field may be applied, in combination with control of the flowof liquid 555 surrounding fluidic droplet 550, that causes the dropletto impact junction 561, which may cause the droplet to split into twoseparate fluidic droplets 565, 566. Fluidic droplet 565 is directed tochannel 562, while fluidic droplet 566 is directed to channel 566. Ahigh degree of control of the applied electric field may be achieved tocontrol droplet formation; thus, for example, after fluidic droplet 565has been split into droplets 565 and 566, droplets 565 and 566 may be ofsubstantially equal size, or either of droplets 565 and 566 may belarger, e.g., as is shown in FIGS. 8E and 8F, respectively.

As another example, in FIG. 9A, channel 540, carrying fluidic droplet530 and liquid 535, divides into channel 542 and 544. Fluidic droplet530 may be electrically charged, or it may uncharged. Electrode 526 ispositioned near channel 542, while electrode 527 is positioned nearchannel 544. Electrode 528 is positioned near the junction of channels540, 542, and 544. When fluidic droplet 530 reaches the junction, it maybe subjected to an electric field, and/or directed to a channel or otherregion, for example, by directing the surrounding liquid into thechannel. As shown in FIG. 9B, fluidic droplet 530 may be split into twoseparate droplets 565 and 566 by applying an electric field 525 to thedroplet using electrodes 526 and 527. In FIG. 9C, a dipole can beinduced in droplet 530 by applying an electric field 525 to the dropletusing electrodes 527 and 528. Due to the strength of the appliedelectric field, the droplet may be strongly attracted to the right, intochannel 544. Similarly, in FIG. 9D, a dipole may be induced in droplet530 by applying an electric field 525 to the droplet using electrodes526 and 528, causing the droplet to be attracted into channel 542. Bycontrolling which electrodes are used to induce an electric field acrossdroplet 530, and/or the strength of the applied electric field, one ormore fluidic droplets within channel 540 may be sorted and/or split intotwo droplets, and each droplet may independently be sorted and/or split.

Sensing Droplets; Sensing the Content of Droplets

In certain aspects of the invention, sensors are provided that can senseand/or determine one or more characteristics of the fluidic droplets,and/or a characteristic of a portion of the fluidic system containingthe fluidic droplet (e.g., the liquid surrounding the fluidic droplet)in such a manner as to allow the determination of one or morecharacteristics of the fluidic droplets. Characteristics determinablewith respect to the droplet and usable in the invention can beidentified by those of ordinary skill in the art. Non-limiting examplesof such characteristics include fluorescence, spectroscopy (e.g.,optical, infrared, ultraviolet, etc.), radioactivity, mass, volume,density, temperature, viscosity, pH, concentration of a substance, suchas a biological substance (e.g., a protein, a nucleic acid, etc.), orthe like.

In some cases, the sensor may be connected to a processor, which inturn, cause an operation to be performed on the fluidic droplet, forexample, by sorting the droplet, adding or removing electric charge fromthe droplet, fusing the droplet with another droplet, splitting thedroplet, causing mixing to occur within the droplet, etc., for example,as previously described. For instance, in response to a sensormeasurement of a fluidic droplet, a processor may cause the fluidicdroplet to be split, merged with a second fluidic droplet, etc.

One or more sensors and/or processors may be positioned to be in sensingcommunication with the fluidic droplet. “Sensing communication,” as usedherein, means that the sensor may be positioned anywhere such that thefluidic droplet within the fluidic system (e.g., within a channel),and/or a portion of the fluidic system containing the fluidic dropletmay be sensed and/or determined in some fashion. For example, the sensormay be in sensing communication with the fluidic droplet and/or theportion of the fluidic system containing the fluidic droplet fluidly,optically or visually, thermally, pneumatically, electronically, or thelike. The sensor can be positioned proximate the fluidic system, forexample, embedded within or integrally connected to a wall of a channel,or positioned separately from the fluidic system but with physical,electrical, and/or optical communication with the fluidic system so asto be able to sense and/or determine the fluidic droplet and/or aportion of the fluidic system containing the fluidic droplet (e.g., achannel or a microchannel, a liquid containing the fluidic droplet,etc.). For example, a sensor may be free of any physical connection witha channel containing a droplet, but may be positioned so as to detectelectromagnetic radiation arising from the droplet or the fluidicsystem, such as infrared, ultraviolet, or visible light. Theelectromagnetic radiation may be produced by the droplet, and/or mayarise from other portions of the fluidic system (or externally of thefluidic system) and interact with the fluidic droplet and/or the portionof the fluidic system containing the fluidic droplet in such as a manneras to indicate one or more characteristics of the fluidic droplet, forexample, through absorption, reflection, diffraction, refraction,fluorescence, phosphorescence, changes in polarity, phase changes,changes with respect to time, etc. As an example, a laser may bedirected towards the fluidic droplet and/or the liquid surrounding thefluidic droplet, and the fluorescence of the fluidic droplet and/or thesurrounding liquid may be determined. “Sensing communication,” as usedherein may also be direct or indirect. As an example, light from thefluidic droplet may be directed to a sensor, or directed first through afiber optic system, a waveguide, etc., before being directed to asensor.

Non-limiting examples of sensors useful in the invention include opticalor electromagnetically-based systems. For example, the sensor may be afluorescence sensor (e.g., stimulated by a laser), a microscopy system(which may include a camera or other recording device), or the like. Asanother example, the sensor may be an electronic sensor, e.g., a sensorable to determine an electric field or other electrical characteristic.For example, the sensor may detect capacitance, inductance, etc., of afluidic droplet and/or the portion of the fluidic system containing thefluidic droplet.

As used herein, a “processor” or a “microprocessor” is any component ordevice able to receive a signal from one or more sensors, store thesignal, and/or direct one or more responses (e.g., as described above),for example, by using a mathematical formula or an electronic orcomputational circuit. The signal may be any suitable signal indicativeof the environmental factor determined by the sensor, for example apneumatic signal, an electronic signal, an optical signal, a mechanicalsignal, etc.

As a particular non-limiting example, a device of the invention maycontain fluidic droplets containing one or more cells. The cells may beexposed to a fluorescent signal marker that binds if a certain conditionis present, for example, the marker may bind to a first cell type butnot a second cell type, the marker may bind to an expressed protein, themarker may indicate viability of the cell (i.e., if the cell is alive ordead), the marker may be indicative of the state of development ordifferentiation of the cell, etc., and the cells may be directed througha fluidic system of the invention based on the presence/absence, and/ormagnitude of the fluorescent signal marker. For instance, determinationof the fluorescent signal marker may cause the cells to be directed toone region of the device (e.g., a collection chamber), while the absenceof the fluorescent signal marker may cause the cells to be directed toanother region of the device (e.g., a waste chamber). Thus, in thisexample, a population of cells may be screened and/or sorted on thebasis of one or more determinable or targetable characteristics of thecells, for example, to select live cells, cells expressing a certainprotein, a certain cell type, etc.

Definitions

A variety of definitions are now provided which will aid inunderstanding various aspects of the invention. Following, andinterspersed with these definitions, is further disclosure that willmore fully describe the invention. As noted, various aspects of thepresent invention relate to droplets of fluid surrounded by a liquid(e.g., suspended). The droplets may be of substantially the same shapeand/or size, or of different shapes and/or sizes, depending on theparticular application. As used herein, the term “fluid” generallyrefers to a substance that tends to flow and to conform to the outlineof its container, i.e., a liquid, a gas, a viscoelastic fluid, etc.Typically, fluids are materials that are unable to withstand a staticshear stress, and when a shear stress is applied, the fluid experiencesa continuing and permanent distortion. The fluid may have any suitableviscosity that permits flow. If two or more fluids are present, eachfluid may be independently selected among essentially any fluids(liquids, gases, and the like) by those of ordinary skill in the art, byconsidering the relationship between the fluids. The fluids may each bemiscible or immiscible. For example, two fluids can be selected to beessentially immiscible within the time frame of formation of a stream offluids, or within the time frame of reaction or interaction. Where theportions remain liquid for a significant period of time, then the fluidsshould be essentially immiscible. Where, after contact and/or formation,the dispersed portions are quickly hardened by polymerization or thelike, the fluids need not be as immiscible. Those of ordinary skill inthe art can select suitable miscible or immiscible fluids, using contactangle measurements or the like, to carry out the techniques of theinvention.

As used herein, a first entity is “surrounded” by a second entity if aclosed planar loop can be drawn around the first entity through only thesecond entity. A first entity is “completely surrounded” if closed loopsgoing through only the second entity can be drawn around the firstentity regardless of direction (orientation of the loop). In oneembodiment, the first entity is a cell, for example, a cell suspended inmedia is surrounded by the media. In another embodiment, the firstentity is a particle. In yet another embodiment, the first entity is afluid. The second entity may also be a fluid in some cases (e.g., as ina suspension, an emulsion, etc.), for example, a hydrophilic liquid maybe suspended in a hydrophobic liquid, a hydrophobic liquid may besuspended in a hydrophilic liquid, a gas bubble may be suspended in aliquid, etc. Typically, a hydrophobic liquid and a hydrophilic liquidare essentially immiscible with respect to each other, where thehydrophilic liquid has a greater affinity to water than does thehydrophobic liquid. Examples of hydrophilic liquids include, but are notlimited to, water and other aqueous solutions comprising water, such ascell or biological media, salt solutions, etc., as well as otherhydrophilic liquids such as ethanol. Examples of hydrophobic liquidsinclude, but are not limited to, oils such as hydrocarbons, siliconeoils, mineral oils, fluorocarbon oils, organic solvents etc. Otherexamples of suitable fluids have been previously described.

Similarly, a “droplet,” as used herein, is an isolated portion of afirst fluid that is completely surrounded by a second fluid. It is to benoted that a droplet is not necessarily spherical, but may assume othershapes as well, for example, depending on the external environment. Inone embodiment, the droplet has a minimum cross-sectional dimension thatis substantially equal to the largest dimension of the channelperpendicular to fluid flow in which the droplet is located.

As mentioned, in some, but not all embodiments, the systems and methodsdescribed herein may include one or more microfluidic components, forexample, one or more microfluidic channels. “Microfluidic,” as usedherein, refers to a device, apparatus or system including at least onefluid channel having a cross-sectional dimension of less than 1 mm, anda ratio of length to largest cross-sectional dimension of at least 3:1.A “microfluidic channel,” as used herein, is a channel meeting thesecriteria. The “cross-sectional dimension” of the channel is measuredperpendicular to the direction of fluid flow within the channel. Thus,some or all of the fluid channels in microfluidic embodiments of theinvention may have maximum cross-sectional dimensions less than 2 mm,and in certain cases, less than 1 mm. In one set of embodiments, allfluid channels containing embodiments of the invention are microfluidicor have a largest cross sectional dimension of no more than 2 mm or 1mm. In certain embodiments, the fluid channels may be formed in part bya single component (e.g. an etched substrate or molded unit). Of course,larger channels, tubes, chambers, reservoirs, etc. can be used to storefluids and/or deliver fluids to various components or systems of theinvention. In one set of embodiments, the maximum cross-sectionaldimension of the channel(s) containing embodiments of the invention isless than 500 microns, less than 200 microns, less than 100 microns,less than 50 microns, or less than 25 microns.

A “channel,” as used herein, means a feature on or in an article(substrate) that at least partially directs flow of a fluid. The channelcan have any cross-sectional shape (circular, oval, triangular,irregular, square or rectangular, or the like) and can be covered oruncovered. In embodiments where it is completely covered, at least oneportion of the channel can have a cross-section that is completelyenclosed, or the entire channel may be completely enclosed along itsentire length with the exception of its inlet(s) and/or outlet(s). Achannel may also have an aspect ratio (length to average cross sectionaldimension) of at least 2:1, more typically at least 3:1, 5:1, 10:1,15:1, 20:1, or more. An open channel generally will includecharacteristics that facilitate control over fluid transport, e.g.,structural characteristics (an elongated indentation) and/or physical orchemical characteristics (hydrophobicity vs. hydrophilicity) or othercharacteristics that can exert a force (e.g., a containing force) on afluid. The fluid within the channel may partially or completely fill thechannel. In some cases where an open channel is used, the fluid may beheld within the channel, for example, using surface tension (i.e., aconcave or convex meniscus).

The channel may be of any size, for example, having a largest dimensionperpendicular to fluid flow of less than about 5 mm or 2 mm, or lessthan about 1 mm, or less than about 500 microns, less than about 200microns, less than about 100 microns, less than about 60 microns, lessthan about 50 microns, less than about 40 microns, less than about 30microns, less than about 25 microns, less than about 10 microns, lessthan about 3 microns, less than about 1 micron, less than about 300 nm,less than about 100 nm, less than about 30 nm, or less than about 10 nm.In some cases the dimensions of the channel may be chosen such thatfluid is able to freely flow through the article or substrate. Thedimensions of the channel may also be chosen, for example, to allow acertain volumetric or linear flowrate of fluid in the channel. Ofcourse, the number of channels and the shape of the channels can bevaried by any method known to those of ordinary skill in the art. Insome cases, more than one channel or capillary may be used. For example,two or more channels may be used, where they are positioned inside eachother, positioned adjacent to each other, positioned to intersect witheach other, etc.

In one set of embodiments, the fluidic droplets may contain cells orother entities, such as proteins, viruses, macromolecules, particles,etc. As used herein, a “cell” is given its ordinary meaning as used inbiology. The cell may be any cell or cell type. For example, the cellmay be a bacterium or other single-cell organism, a plant cell, or ananimal cell. If the cell is a single-cell organism, then the cell maybe, for example, a protozoan, a trypanosome, an amoeba, a yeast cell,algae, etc. If the cell is an animal cell, the cell may be, for example,an invertebrate cell (e.g., a cell from a fruit fly), a fish cell (e.g.,a zebrafish cell), an amphibian cell (e.g., a frog cell), a reptilecell, a bird cell, or a mammalian cell such as a primate cell, a bovinecell, a horse cell, a porcine cell, a goat cell, a dog cell, a cat cell,or a cell from a rodent such as a rat or a mouse. If the cell is from amulticellular organism, the cell may be from any part of the organism.For instance, if the cell is from an animal, the cell may be a cardiaccell, a fibroblast, a keratinocyte, a heptaocyte, a chondracyte, aneural cell, a osteocyte, a muscle cell, a blood cell, an endothelialcell, an immune cell (e.g., a T-cell, a B-cell, a macrophage, aneutrophil, a basophil, a mast cell, an eosinophil), a stem cell, etc.In some cases, the cell may be a genetically engineered cell. In certainembodiments, the cell may be a Chinese hamster ovarian (“CHO”) cell or a3T3 cell.

Materials

A variety of materials and methods, according to certain aspects of theinvention, can be used to form any of the above-described components ofthe systems and devices of the invention. In some cases, the variousmaterials selected lend themselves to various methods. For example,various components of the invention can be formed from solid materials,in which the channels can be formed via micromachining, film depositionprocesses such as spin coating and chemical vapor deposition, laserfabrication, photolithographic techniques, etching methods including wetchemical or plasma processes, and the like. See, for example, ScientificAmerican, 248:44-55, 1983 (Angell, et al). In one embodiment, at least aportion of the fluidic system is formed of silicon by etching featuresin a silicon chip. Technologies for precise and efficient fabrication ofvarious fluidic systems and devices of the invention from silicon areknown. In another embodiment, various components of the systems anddevices of the invention can be formed of a polymer, for example, anelastomeric polymer such as polydimethylsiloxane (“PDMS”),polytetrafluoroethylene (“PTFE” or Teflon®, or the like.

Different components can be fabricated of different materials. Forexample, a base portion including a bottom wall and side walls can befabricated from an opaque material such as silicon or PDMS, and a topportion can be fabricated from a transparent or at least partiallytransparent material, such as glass or a transparent polymer, forobservation and/or control of the fluidic process. Components can becoated so as to expose a desired chemical functionality to fluids thatcontact interior channel walls, where the base supporting material doesnot have a precise, desired functionality. For example, components canbe fabricated as illustrated, with interior channel walls coated withanother material. Material used to fabricate various components of thesystems and devices of the invention, e.g., materials used to coatinterior walls of fluid channels, may desirably be selected from amongthose materials that will not adversely affect or be affected by fluidflowing through the fluidic system, e.g., material(s) that is chemicallyinert in the presence of fluids to be used within the device.

In one embodiment, various components of the invention are fabricatedfrom polymeric and/or flexible and/or elastomeric materials, and can beconveniently formed of a hardenable fluid, facilitating fabrication viamolding (e.g. replica molding, injection molding, cast molding, etc.).The hardenable fluid can be essentially any fluid that can be induced tosolidify, or that spontaneously solidifies, into a solid capable ofcontaining and/or transporting fluids contemplated for use in and withthe fluidic network. In one embodiment, the hardenable fluid comprises apolymeric liquid or a liquid polymeric precursor (i.e. a “prepolymer”).Suitable polymeric liquids can include, for example, thermoplasticpolymers, thermoset polymers, or mixture of such polymers heated abovetheir melting point. As another example, a suitable polymeric liquid mayinclude a solution of one or more polymers in a suitable solvent, whichsolution forms a solid polymeric material upon removal of the solvent,for example, by evaporation. Such polymeric materials, which can besolidified from, for example, a melt state or by solvent evaporation,are well known to those of ordinary skill in the art. A variety ofpolymeric materials, many of which are elastomeric, are suitable, andare also suitable for forming molds or mold masters, for embodimentswhere one or both of the mold masters is composed of an elastomericmaterial. A non-limiting list of examples of such polymers includespolymers of the general classes of silicone polymers, epoxy polymers,and acrylate polymers. Epoxy polymers are characterized by the presenceof a three-membered cyclic ether group commonly referred to as an epoxygroup, 1,2-epoxide, or oxirane. For example, diglycidyl ethers ofbisphenol A can be used, in addition to compounds based on aromaticamine, triazine, and cycloaliphatic backbones. Another example includesthe well-known Novolac polymers. Non-limiting examples of siliconeelastomers suitable for use according to the invention include thoseformed from precursors including the chlorosilanes such asmethylchlorosilanes, ethylchlorosilanes, phenylchlorosilanes, etc.

Silicone polymers are preferred in one set of embodiments, for example,the silicone elastomer polydimethylsiloxane. Non-limiting examples ofPDMS polymers include those sold under the trademark Sylgard by DowChemical Co., Midland, Mich., and particularly Sylgard 182, Sylgard 184,and Sylgard 186. Silicone polymers including PDMS have severalbeneficial properties simplifying fabrication of the microfluidicstructures of the invention. For instance, such materials areinexpensive, readily available, and can be solidified from aprepolymeric liquid via curing with heat. For example, PDMSs aretypically curable by exposure of the prepolymeric liquid to temperaturesof about, for example, about 65° C. to about 75° C. for exposure timesof, for example, about an hour. Also, silicone polymers, such as PDMS,can be elastomeric and thus may be useful for forming very smallfeatures with relatively high aspect ratios, necessary in certainembodiments of the invention. Flexible (e.g., elastomeric) molds ormasters can be advantageous in this regard.

One advantage of forming structures such as microfluidic structures ofthe invention from silicone polymers, such as PDMS, is the ability ofsuch polymers to be oxidized, for example by exposure to anoxygen-containing plasma such as an air plasma, so that the oxidizedstructures contain, at their surface, chemical groups capable ofcross-linking to other oxidized silicone polymer surfaces or to theoxidized surfaces of a variety of other polymeric and non-polymericmaterials. Thus, components can be fabricated and then oxidized andessentially irreversibly sealed to other silicone polymer surfaces, orto the surfaces of other substrates reactive with the oxidized siliconepolymer surfaces, without the need for separate adhesives or othersealing means. In most cases, sealing can be completed simply bycontacting an oxidized silicone surface to another surface without theneed to apply auxiliary pressure to form the seal. That is, thepre-oxidized silicone surface acts as a contact adhesive againstsuitable mating surfaces. Specifically, in addition to beingirreversibly sealable to itself, oxidized silicone such as oxidized PDMScan also be sealed irreversibly to a range of oxidized materials otherthan itself including, for example, glass, silicon, silicon oxide,quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, andepoxy polymers, which have been oxidized in a similar fashion to thePDMS surface (for example, via exposure to an oxygen-containing plasma).Oxidation and sealing methods useful in the context of the presentinvention, as well as overall molding techniques, are described in theart, for example, in an article entitled “Rapid Prototyping ofMicrofluidic Systems and Polydimethylsiloxane,” Anal. Chem., 70:474-480,1998 (Duffy et al.), incorporated herein by reference.

Another advantage to forming microfluidic structures of the invention(or interior, fluid-contacting surfaces) from oxidized silicone polymersis that these surfaces can be much more hydrophilic than the surfaces oftypical elastomeric polymers (where a hydrophilic interior surface isdesired). Such hydrophilic channel surfaces can thus be more easilyfilled and wetted with aqueous solutions than can structures comprisedof typical, unoxidized elastomeric polymers or other hydrophobicmaterials.

In one embodiment, a bottom wall is formed of a material different fromone or more side walls or a top wall, or other components. For example,the interior surface of a bottom wall can comprise the surface of asilicon wafer or microchip, or other substrate. Other components can, asdescribed above, be sealed to such alternative substrates. Where it isdesired to seal a component comprising a silicone polymer (e.g. PDMS) toa substrate (bottom wall) of different material, the substrate may beselected from the group of materials to which oxidized silicone polymeris able to irreversibly seal (e.g., glass, silicon, silicon oxide,quartz, silicon nitride, polyethylene, polystyrene, epoxy polymers, andglassy carbon surfaces which have been oxidized). Alternatively, othersealing techniques can be used, as would be apparent to those ofordinary skill in the art, including, but not limited to, the use ofseparate adhesives, thermal bonding, solvent bonding, ultrasonicwelding, etc.

The following examples are intended to illustrate certain embodiments ofthe present invention, but do not exemplify the full scope of theinvention.

EXAMPLE 1

FIG. 15A illustrates an example of a fluidic system having some of thefeatures described above. In this example, fluidic droplets areintroduced into fluidic system 800 (shown schematically in FIG. 15A.Fluidic system 800 comprises an inlet region 801 (labeled “a”), abifurcation region 802 (“b”), a region comprising posts 803 (“c”), and acollection region 804 (“d”). Inlet region 801 produces a series ofdroplets 810 contained within liquid 815. The droplets have an averagediameter of about 20 micrometers. The liquid, in this example, is water,and the fluidic droplets comprise hexadecane with about 3 wt % SPAN80 (asurfactant). The series of droplets are illustrated in FIG. 15B, whichis an enlargement of inlet region 801 shown schematically in FIG. 15A.

In FIG. 15C, a series of bifurcations (shown to the left of thephotomicrograph) divide the fluidic droplets into a series of channels.Since the droplets are not purposefully directed towards any particularchannel, the droplets are randomly dispersed within the channels. Thedroplets are then carried to a region comprising a series of posts 818.An enlargement of this region is illustrated in FIG. 15D. As can beseen, the individual droplets maintain their individual identities anddo not fuse, due to the presence of the surfactant. In FIG. 15E, thedroplets are collected in collection region 804.

EXAMPLE 2

In this example, precision manipulation of streams of fluids with amicrofluidic device is demonstrated. This technology enables highthroughput reactors that use minute quantities of reagents. As the scaleof these reactors shrinks, contamination effects due to surfaceadsorption and diffusion may restrict the smallest quantities that canbe used. Confinement of reagents in droplets in an immiscible carrierfluid may overcome these limitations, but demands new fluid-handlingtechnology. An example platform technology is presented based on chargeddroplets and electric fields that enables electrically addressabledroplet generation, highly efficient droplet coalescence, precisiondroplet breaking and recharging, and controllable droplet sorting.

In this example, a generic and robust platform technology is presentedfor manipulating and controlling individual droplets in a microfluidicdevice. By combining electrostatic charge on the droplets and electricfields on the devices modules that create, recombine, split, and sortdroplets individually are illustrated, providing exquisite control overindividual microreactors while retaining high purity and enabling veryhigh throughput. By incorporating the forces that result from chargingthe aqueous fluid in an electric field, E, smaller droplets are producedwith more precise control of their individual timing than is feasiblewith other strategies that rely solely on viscous forces to overcomesurface tension; this provides a robust droplet generation module thatallows the production of microreactors with volumes as small asfemtoliters. Incorporating charge of opposite signs on differentdroplets allows droplets to be controllably and reliably merged,overcoming the stabilizing forces due to surface tension andlubrication; this provides a robust droplet coalescence module thatallows the precise mixing of aliquots of reactants. By incorporating theextensional force induced by an electric field large droplets may becontrollably split into smaller aliquots for further analysis, and insome cases, it may simultaneously recharge neutral droplets for furtherprocessing; this provides a robust splitting or charging module thatallows multiple assays to be performed on the same materials. Byincorporating the forces produced by electric fields on chargeddroplets, individual droplets may be steered into selected channels;this provides a robust droplet sorting module that allows desiredreaction products to be selected. These modules are useful for highspeed manipulation and control of individual droplets, and can serve asthe technology for droplet-based microfluidic devices. Moreover, becauseall control is achieved by switching electric fields, there are nomoving parts and frequencies as high as 106 Hz are feasible; thisfacilitates very high throughput combinatorial technology.

Soft lithography was used to pattern channels in polydimethylsiloxane(PDMS), a transparent polymer material. A glass slide forms the top ofthe channel. Electric fields were incorporated by patterningindium-tin-oxide (ITO) electrodes on the surface of the glass slideadjacent to the channels and seal the slide to the PDMS using an oxygenplasma. Devices fabricated in PDMS have the advantage of being stronglyhydrophobic ensuring that the oil carrier phase wets their surfaces andthat the water droplets do not contact the walls of the channel walls,facilitating the isolation of biomolecules and eliminating crosscontamination due to surface interactions.

A flow-focusing geometry was used to form the droplets. A water streamwas infused from one channel through a narrow constriction; counterpropagating oil streams hydrodynamically focus the water stream reducingits size as it passes through the constriction. This droplet generatorcan be operated in a flow regime that produces a steady stream ofuniform droplets of water in oil. The size of the water droplets wascontrolled by the relative flow rates of the oil and the water; theviscous forces overcome surface tension to create uniform drops. If theflow rate of the water was too high a longer jet of fluid passes throughthe orifice and breaks up into droplets further downstream; thesedroplets were less uniform in size. If the flow rate of the water wastoo low, the droplet breakup in the orifice becomes irregular, producinga wider range of droplet sizes.

Electric fields were then incorporated to create an electricallyaddressable emulsification system. To achieve this, high voltage wasapplied to the aqueous stream and charge the oil water interface. Thewater stream behaved as a conductor while the oil was an insulator;electrochemical reactions charged the fluid interface like a capacitor.At snap-off, the charge on the interface remains on the droplet. Inaddition, the droplet volume, Vd, and frequency, f, could be tailoredover at least three orders of magnitude without changing the infusionrate of the oil or water. Droplet size and frequency were notindependent in this example; instead their product is determined by theinfusion rate of the dispersed phase Qd=fVd. The droplet size decreasedwith increasing field strength. At low field strength, the droplet sizewas determined by the flowrate of the continuous phase. However, at highfield strength, droplet size was determined by the electric field anddecreased rapidly with E.

The dependence of the droplet size on applied voltage for threedifferent flow rates is as follows. At low applied voltages the electricfield had a negligible effect, and droplet formation was driven by thecompetition between surface tension and viscous flow. By contrast, athigh electric field strengths, there was a significant additional forceon the growing drop, F=qE, where q is the charge on the droplet. Sincethe droplet interface behaved as a capacitor, q is proportional to theapplied voltage, V. This led to a V 2 dependence of the force, whichaccounted for the decrease in droplet size with increasing appliedfield. For even higher electric fields, the charged interface of thewater stream was repelled by the charged drops.

In one embodiment, oil and water streams converge at 30 micron orifice.A voltage V applied to indium-tin-oxide (ITO) electrodes on the glassproduced an electric field E to capacitively charge the aqueous-oilinterface. Droplet size was found to be independent of charge at lowfield strengths but decreased at higher fields. Droplet size is afunction of voltage, showing the crossover between flow-dominated andfield-dominated snap-off for three different flow rates of thecontinuous phase oil (Qc=80 nL/s, 110 nL/s, and 140 nL/s). The infusionrate of the water was constant at Qd=20 nL/s.

The electronic control afforded by the field-induced droplet formationprovides an additional benefit in this example: it allowed the phase ofthe droplet break-off to be adjusted within the production cycle. Thiswas accomplished by increasing the field above the critical break-offfield only at the instant the droplet is required. This provided aconvenient means to precisely synchronize the production and arrival ofindividual droplets in specific locations.

An important component in some droplet-based reaction confinement systemis a mixer which combines two or more reagents to initiate a chemicalreaction. An example of a mixer uses electrostatic charges; placingcharges of opposite sign on each droplet and applying an electric fieldcauses them to coalesce. As an example, a device is illustrated havingtwo separate nozzles that generate droplets with different compositionsand opposite charges. Droplets were brought together at the confluenceof the two streams. The electrodes used to charge the droplets uponformation also provide the electric field to force the droplets acrossthe stream lines, leading to coalesce. Slight variations in thestructure of the two nozzles resulted in slight differences in thefrequency and phase of their droplet generation in the absence of afield. Thus the droplets differed in size, even though the infusionrates were identical. Moreover, the droplets did not arrive at the pointof confluence at exactly the same time. As a result the droplets did notcoalesce. By contrast, upon application of an electric field, dropletformation became generally synchronized, ensuring that pairs ofidentically sized droplets reached the point of confluencesimultaneously. Moreover, the droplets were oppositely charged, causingthem to traverse the stream lines and contact each other thereby causingthem to coalesce. The synchronization of droplet formation resulted fromcoupling of the break-off of the two droplets as mediated by theelectric field; the magnitude of the electric field varied as theseparation between the leading edges of the two droplets changes and thefrequency of droplet break-off is mode locked to the electric field. Aminimum charge is required to cause droplets to coalesce in thisexample, presumably because of the stabilizing effects of the surfactantcoating; the E field depends on the percentage of droplets thatcontacted each other that actually coalesce.

In one embodiment, droplets having opposite sign of electrostatic chargecan be generated by applying a voltage across the two aqueous streams.In another embodiment, in the absence of the field, the frequency andtiming of droplet formation at the two nozzles may be independent, andeach nozzle may produce a different size droplet at a differentfrequency; infusion rates are the same at both nozzles. After theconfluence of the two streams, droplets from the upper and lower nozzlesstay in their respective halves of the stream. Due to surfactant, thereare no coalescence events even in the case of large slugs that fill thechannel width. In yet another embodiment, with an applied voltage of200V across the 500 micron separation of the nozzles, the droplets thatsimultaneously break-off from the two nozzles are essentially identical;simultaneous droplet formation can be achieved for unequal infusionrates of the aqueous streams even up to a factor of two difference involumes. The fraction of the droplets that encounter each other- andcoalesce increases linearly above a critical field when a surfactant,sorbiton-monooleate 3% is present.

The use of oppositely charged droplets and an electric field to combineand mix reagents was extremely robust, and nearly 100% of the dropletsfrom the two streams coalesced with their partners from the oppositestream. However, after they coalesced the resultant droplets carriedessentially no electrostatic charge. While it is convenient to chargedroplets during formation, other methods may be employed in any robustdroplet-based microfluidic system to recharge the-mixed droplets, ifnecessary, for further processing. This may be accomplished, forexample, through the use of extensional flow to split neutral dropletsin the presence of an electric field which polarizes the dropletsresulting in two oppositely charged daughter droplets. In oneembodiment, neutral droplets enter a bifurcation and split into chargeddaughter droplets. In some cases, the asymmetric stretching of thecharged droplets in the electric field can be observed. The verticaldashed lines indicate the edge of the electrodes where the dropletsreturned to their symmetric spherical shape. The electric field alsoallowed precision control of the droplet splitting, providing the basisof a robust droplet division module which allows the splitting of thecontents into two or more aliquots of identical reagent facilitatingmultiple assays on the contents of the same microreactor.

In another embodiment, neutral droplets can be recharged by breakingthem in the presence of an electric field. Uncharged droplets (q=0) werepolarized in an electric field (ES not equal to 0), provided ES wassufficiently large, and the droplets break into two oppositely chargeddaughter drops in the extensional flow at a bifurcation. The chargeddroplets were stretched in the electric field ES, but returned tospherical on contacting the electrodes.

Another component useful for the construction of microfluidic dropletreaction systems is a droplet sorter. The contents of individual maymust be probed, and selected droplets may be sorted into discreetstreams. Such sorting in microfluidic devices can be accomplished, asshown in this example, through the use of mechanical valves. The use ofelectrostatic charging of droplets may provide an alternate means thatcan be precisely controlled, can be switched at high frequencies, andrequires no moving parts. Electrostatic charge on the droplets mayenable drop-by-drop sorting based on the linear coupling of charge to anexternal electric field. A T-junction bifurcation that splits the flowof carrier fluid equally will also randomly split the droplet populationequally into the two streams. However, a small electric field applied atthe bifurcation may precisely dictate which channel the droplets enter;varying the direction of the field varies the direction of the dropletsorting. The large forces that can be imparted on the droplets and thehigh switching frequency make this a fast and robust sorting engine withno moving parts; thus the processing rate is limited primarily by therate of droplet generation.

In one embodiment, charged droplets alternately entered the right andleft channels when there was no field applied (ES=0). When an electricfield is applied to the right, the droplets entered the right branch atthe bifurcation; they entered the left branch when the field isreversed. After the bifurcation, the distance between droplets isreduced to half what it was before, indicating the oil stream is evenlydivided. In some cases, the deformation in the shape of a highly chargeddrop in an electric field can be observed.

The enhanced functionality that electrostatic charge brings to dropletsin microfluidic devices enables an expansive list of microfluidicsapplications. This toolkit of techniques for manipulating droplets willenable modular integration of systems for transporting and reactingsmall numbers of molecules. High throughput screening, combinatorialchemistry, and the search for rare biological function in librariescould all potentially benefit from electrostatic manipulation ofdroplets in microchannels. For instance, droplet based microfluidictechnology can also be used to develop a chip-scale fluorescenceactivated cell sorter (FACS) with enhanced activation functionality thatgoes beyond fluorescence to include multiple reagent-based assaysbetween the droplet formation and sorting steps. Moreover by usingfemtoliter drops, which are a few microns in diameter, even a singlebiomolecule represents a concentrations of >>1 nM, sufficient forefficient chemical reactivity and single molecule assays.

Many of the uses of droplet-based microfluidic devices are driven by aneed to encapsulate a varied population or library of molecules, cellsor particles into microreactors, perform an assay on the contents,perhaps through the addition of reagents, and then, finally, toselectively remove specific microreactors from the collection in asearch for rare events. This requires a processing rate of 103 persecond to sort through the smallest libraries in a reasonable time whilerates on the order 105 per second are desirable for larger libraries.These rates are feasible, as discussed herein. Moreover, because themicrofluidic devices may be produced using stamping techniques, e.g., asdescribed herein, parallel flow streams or fluidic systems can befabricated, further enhancing the total throughput. Combined, theadvantages of droplets and high throughput manipulation providesignificant opportunity for widespread application.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of”, when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing” “involving,” “holding,”“composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

1. A method of combining at least two species in a controlled manner ina microfluidic system, comprising: providing a series of dropletsflowing in a microfluidic system; selecting a first droplet from theseries of droplets and separating the first droplet from at least someother droplets in the series of droplets, wherein the first droplet hasa maximum cross-sectional dimension of less than about 100 microns andcontains a first chemical, biological, or biochemical species; providinga second droplet separate from the series of droplets, wherein thesecond droplet has a maximum cross-sectional dimension of less thanabout 100 microns and contains a second chemical, biological, orbiochemical species; selectively urging the first droplet and/or thesecond droplet toward a location where coalescence can occur, andallowing the first droplet and the second droplet to coalesce into onecombined droplet; and determining a reaction involving at least thefirst species in the first droplet and the second species in the seconddroplet.
 2. A method of sorting droplets in a controlled manner in amicrofluidic system, comprising: providing a series of droplets flowingin a microfluidic system; and selecting a first droplet from the seriesof droplets and separating the first droplet from at least some otherdroplets in the series of droplets, wherein the first droplet has amaximum cross-sectional dimension of less than about 100 microns.
 3. Amethod as in claim 2, further comprising selecting a second droplet fromthe series of droplets and separating the second droplet from at leastsome other droplets in the series of droplets, wherein the seconddroplet has a maximum cross-sectional dimension of less than about 100microns.
 4. A method as in claim 2, wherein the first droplet contains achemical, biological, or biochemical species other than the fluiddefining the droplet.
 5. A method as in claim 2, wherein the firstdroplet does not contain a chemical, biological, or biochemical speciesother than the fluid defining the droplet.
 6. A method as in claim 2,comprising sorting a plurality of the droplets flowing in themicrofluidic system by determining, for each of the plurality ofdroplets, an amount or lack of a contained specific chemical,biological, or biochemical species and if the droplet contains a firstamount of or does not contain the species directing the droplet into afirst channel and if the droplet contains a second amount of the speciesdirecting the droplet into a second channel. 7-10. (canceled)
 11. Amethod of combining at least two droplets in a microfluidic system,comprising: providing at least two droplets in a microfluidic system;exposing the droplets to an electric field thereby inducing, in thedroplets, dipole moments; and coalescing the at least two droplets intoa single droplet at least in part via droplet-droplet attraction due tothe induced dipole moments.
 12. A method as in claim 11, comprisingcoalescing the droplets under conditions in which, in the absence of thefield-induced dipole moments, the droplets would not coalesce. 13-273.(canceled)
 274. A method as in claim 1, wherein at least about 90% ofthe series of droplets each consists of the same number of entities ofthe first chemical, biological, or biochemical species therein.
 275. Themethod of claim 1, wherein the series of droplets have an averagediameter that is less than about 200 micrometers.
 276. A method as inclaim 1, wherein the act of allowing the first droplet and the seconddroplet to coalesce into one combined droplet comprises inducing apositive charge on the first droplet and a negative charge on the seconddroplet.
 277. A method as in claim 276, wherein the positive charge onthe first droplet is at least about 10⁻¹⁶ C.
 278. A method as in claim2, wherein the act of selecting occurs at a rate of at least about 10droplets/s.
 279. A method as in claim 278, wherein the act of selectingoccurs at a rate of at least about 1000 droplets/s.
 280. A method as inclaim 279, wherein the act of selecting occurs at a rate of at leastabout 100,000 droplets/s.
 281. A method as in claim 2, comprisingselecting the first droplet based on a characteristic of the firstdroplet.
 282. A method as in claim 2, wherein the first dropletcomprises a cell.
 283. A method as in claim 11, wherein the electricfield has an intensity of at least about 0.01 V/micrometer.
 284. Amethod as in claim 11, wherein the at least two droplets havesubstantially the same volume.
 285. A method as in claim 11, whereineach of the at least two droplets have an average diameter that is lessthan about 200 micrometers.